The overarching objective of this work is to gain a comprehensive understanding of the nucleic acid (NA) chaperone function of the HIV nucleocapsid (NC) protein and the Gag polyprotein precursor. Many of NC's functions rely on its chaperone activity, i.e., the ability to catalyze NA conformational rearrangements that lead to the most thermodynamically stable structure. The impact of this work is high due to NC's role in almost every stage of the viral lifecycle. NC's NA binding and chaperone function has been demonstrated to play an important role in reverse transcription, integration, RNA packaging, and viral assembly, and these studies will address open questions in our molecular level understanding of many of these processes. During the previous grant period, using biochemical assays and ensemble and single molecule biophysical approaches, we gained novel insights into the mechanism by which HIV NC facilitates NA rearrangements. We also initiated studies of NC in the context of HIV Gag. We discovered that Gag's chaperone activity requires the NC domain and surprisingly, is stimulated by inositol phosphate (IP) binding to the matrix (MA) domain. We will continue to employ innovative biophysical and biochemical approaches to improve our understanding of the mechanism of NC's chaperone activity, and will expand our studies to investigate in detail the relatively poorly understood chaperone function of NC in the context of Gag. We are particularly interested in the mechanism by which HIV MA modulates Gag's chaperone properties. The specific aims are: (1) To probe the NA chaperone activity of WT, mutant and precursor forms of HIV-1 NC, and (2) to probe HIV-1 Gag's chaperone activity in vitro and in vivo. PUBLIC HEALTH RELEVANCE: The HIV nucleocapsid protein (NC) is a "chaperone" protein that facilitates refolding of nucleic acids (DNA and RNA) during the retroviral lifecycle. The remarkable nucleic acid chaperone properties of NC, its high genetic barrier to mutation, and its central role in multiple stages of retrovirus replication make NC an especially attractive target for new HIV therapeutics. The overarching objective of this work, to contribute to our understanding of the nucleic acid binding and chaperone functions of HIV NC, may lead to new strategies for targeting this essential retroviral protein.